DNA molecules encoding the melanocortin 5 receptor protein from rhesus monkey

ABSTRACT

The present invention relates to rhesus monkey DNA molecules encoding the melanocortin-5 receptor protein, recombinant vectors comprising DNA molecules encoding rhesus MC-5R, recombinant host cells which contain a recombinant vectors encoding rhesus MC-5R, the rhesus MC-5R protein encoded by the DNA molecule, and methods of identifying selective agonists and antagonists of rhesus MC-5R.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Ser. No. 60/107,632,filed Nov. 9, 1998, which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to rhesus monkey (Macaca mulatta) DNAmolecules encoding the melanocortin-5 receptor protein belonging to therhodopsin sub-family of G-protein coupled receptors, recombinant vectorscomprising DNA molecules encoding rhesus MC-5R, recombinant host cellswhich contain a recombinant vector encoding rhesus MC-5R, the rhesusMC-5R protein encoded by the DNA molecule, and methods of identifyingselective agonists and antagonists of rhesus MC-5R.

BACKGROUND OF THE INVENTION

Melanocortin receptors belong to the rhodopsin sub-family of G-proteincoupled receptors (GPCR's). Five different subtypes are known. Thesemelanocortin receptors bind and are activated by peptides such as α-, β,or γ-melanocyte stimulating hormones (α-, β-, γ-MSH) derived from thepro-opiomelanocortin (POMC) gene. A wide range of physiologicalfunctions are believed to be mediated by melanocortin peptides and theirreceptors.

U.S. Pat. No.5,532,347, issued on Jul. 2, 1996, to Cone and Mountjoydiscloses human and mouse DNA molecules which encode MC-1R (also knownin the art as α-MSH-R). The expressed human protein contains 317 aminoacids.

U.S. Pat. No. 5,280,112 (issued Jan. 18, 1994) and U.S. Pat.No.5,554,729 (issued Sep. 10, 1996), both to Cone and Mountjoy, disclosehuman and mouse DNA molecules which encode MC-2R (also known in the artas ACTH-R). The human MC-2R protein contains 297 amino acids.

Mountjoy, et al. (1992, Science 257: 1248-1251) describe DNA moleculesand the concomitant protein for human MC-1R and human MC-2R.

Chhajlani, et al. (1992, FEBS Letters 309: 417-420) also disclose ahuman DNA molecule comprising an open reading frame which encodes humanMC1-R.

Roselli-Rehfuss, et al, (1993, Proc. Natl. Acad. Sci 90: 8856-8860)disclose a cDNA clone encoding rat MC-3R cDNA.

U.S. Pat. No. 5,622,860 (issued Apr. 22, 1997) and U.S. Pat. No.5,703,220 (issued Dec. 30, 1997) to Yamada and Gantz, disclose DNAmolecules which encode human MC-3R and human MC-4R, respectively (seealso Gantz, et al., 1993, J. Biol. Chem. 268(11): 8246-8250).

A DNA molecule encoding human MC-5R was also disclosed by Mountjoy, etal. (1994, Mol. Endocrin. 8: 1298-1308).

Chhajlani, et al. (1993, Biochem. Biophys. Res. Comm., 195(2): 866-873)disclose a DNA molecule which the authors state encodes MC-5R. Thisclone was initially designated MC2.

Fathi, et al. (1995, Neurochenrical Research 20(1):107-113) alsodisclose a DNA molecule thought to encode human MC-5R. There are severalsequence discrepancies when compared to the DNA molecule disclosed byChhajlani, et al., id.

Griffon, et al. (1994, Biochem. Biophys. Res. Comm., 200(2): 1007-1014)disclose DNA clones from human and rat which encode MC-5R. The human DNAsequence agrees with the human DNA sequence disclosed in Fathi et al.id.

Gantz, et al. (1994, Biochem. Biophys. Res. Comm., 200(3): 11214-11220;see also U.S. Pat. No. 5,710,265, issued Jan. 20, 1998 to Yamada andGantz) and Labbe, et al. (1994, Biochemistry 33: 4543-4549) disclose DNAclones from mouse which encode MC-5R.

Barrett, et al. (1994, J. Mol. Endocrin. 12: 203-213) disclose DNAclones from sheep which encode MC-5R.

In rodents, MC-4R has been implicated as a key regulator of feedingbehavior which regulates body weight through studies with peptideagonists and antagonists (Fan et al., 1997, Nature 385: 165-168) andwith a MC-4R knock-out mouse (Huszar et al., 1997, Cell 88: 131-141).

Compounds that bind to such receptors were previously identified bybinding to human and/or rodent receptors and evaluated for theirefficacy in rodents. However, the neuroendocrine process can differbetween rodents and man. It is also expected that some compounds exhibitdifferent binding affinities for different species homologues of thesame receptor (Fong et al., 1992, J. Biol. Chem. 267:25666-25671; Hartiget al., 1992, TIPS 13:152-159).

Before compounds can be selected as a drug candidate it is firstevaluated for a physiological effect in rodents and then in the rhesusprimate. It is often that one compound may be effective in one animalspecies but not in another. Previously, it has been impossible todetermine if the failure was due to an altered melanocortin pathway indifferent species, or due to a compound's having a lower affinity forone particular species. Past protocols required the use of a rhesusbrain membrane to determine the in vitro biochemical activity ofcompounds, if such protocol could be successfully employed.

It is desirable to correlate in vivo data with in vitro biochemicalactivity of compounds.

It is also desirable to first select compounds that are active for therhesus receptor in vitro.

It is also desirable to identify compounds which can determine therelevance of receptor targets in rhesus and allow selection of noveldrugs to treat obesity.

It is further desirable to discover new drugs which effectpathophysiological processes by modulating the effects in rhesus toidentify melanocortin active process in primates, followed by humanclinical trials.

The present invention addresses and meets these needs by disclosing anisolated nucleic acid fragment which expresses a form of rhesus MC-5R,recombinant vectors which house this nucleic acid fragment, recombinanthost cells which expresses rhesus MC-5R and/or a biologically activeequivalent, and pharmacological properties of this rhesus MC-5R protein.

SUMMARY OF THE INVENTION

The present invention relates to an isolated nucleic acid molecule(polynucleotide) which encodes a novel rhesus monkey (Macaca mulatta)melanocortin-5 receptor (rhMC-5R). The nucleic acid molecules of thepresent invention are substantially free from other nucleic acids.

The present invention relates to an isolated nucleic acid molecule(polynucleotide) which encodes mRNA which expresses a novel rhesusMC-5R, this DNA molecule comprising the nucleotide sequence disclosedherein as SEQ ID NO:1.

The present invention also relates to biologically active fragments ormutants of SEQ ID NO:1 which encodes mRNA expressing a novel rhesusMC-5R. Any such biologically active fragment and/or mutant will encodeeither a protein or protein fragment which at least substantially mimicsthe pharmacological properties of a wild-type MC-5R protein, includingbut not limited to the rhesus MC-5R receptor truncations andcarboxy-terminal truncations such that these mutations encode mRNA whichexpress a protein or protein fragment of diagnostic, therapeutic orprophylactic use and would be useful for screening for agonists and/orantagonists for MC-5R function.

A preferred aspect of this portion of the present invention is disclosedin FIG. 1, a rhesus cDNA molecule encoding a novel MC-5R (SEQ ID NO:1).

The isolated nucleic acid molecules of the present invention may includea deoxyribonucleic acid molecule (DNA), such as genomic DNA andcomplementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA).

The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, which contain thesubstantially purified nucleic acid molecules disclosed throughout thisspecification.

The present invention also relates to subcellular membrane fractions ofthe recombinant host cells (both prokaryotic and eukaryotic as well asboth stably and transiently transformed cells) which contain theproteins encoded by the nucleic acids of the present invention. Thesesubcellular membrane fractions will comprise either wild-type or mutantforms of rhesus melanocortin-5 receptor proteins at levels substantiallyabove endogenous levels and hence will be useful in various assaysdescribed throughout this specification.

The present invention also relates to a substantially purified form ofthe rhesus melanocortin-5 receptor protein, which comprises the aminoacid sequence disclosed in FIG. 2 and set forth as SEQ ID NO:2. Apreferred aspect of the present invention is disclosed in FIG. 2 and isset forth as SEQ ID NO:2, the amino acid sequence of the novel rhesusmelanocortin-5 receptor protein.

The present invention also relates to biologically active fragmentsand/or mutants of the rhesus melanocortin-5 receptor protein comprisingthe amino acid sequence set forth as SEQ ID NO:2, including but notnecessarily limited to amino acid substitutions, deletions, additions,amino terminal truncations and carboxy-terminal truncations such thatthese mutations provide for proteins or protein fragments of diagnostic,therapeutic or prophylactic use and would be useful for screening foragonists and/or antagonists for MC-5R function.

The present invention also relates to isolated nucleic acid moleculeswhich are fusion constructions expressing fusion proteins useful inassays to identify compounds which modulate wild-type vertebrate MC-5Ractivity. A preferred aspect of this portion of the invention includes,but is not limited to, glutathione S-transferase (GST)-MC-5R fusionconstructs which include, but are not limited to, either theintracellular domain of rhesus MC-5R as an in-frame fusion at thecarboxy terminus of the GST gene, or the extracellular and transmembraneligand binding domain of MC-5R fused to the amino terminus of GST, orthe extracellular and transmembrane domain of MC-5R fused to animmunoglobulin gene by methods known to one of ordinary skill in theart. Soluble recombinant GST-MC-5R fusion proteins may be expressed invarious expression systems, including Spodoptera frugiperda (Sf21)insect cells (Invitrogen) using a baculovirus expression vector (pAcG2T,Phamiingen).

Therefore, the present invention relates to methods of expressing therhesus MC-5R protein and biological equivalents disclosed herein, assaysemploying these gene products, recombinant host cells which comprise DNAconstructs which express these receptor proteins, and compoundsidentified through these assays which act as agonists or antagonists ofMC-5R activity.

The present invention also relates to polyclonal and monoclonalantibodies raised in response to either the rhesus form of MC-5R, or abiologically active fragment thereof.

It is an object of the present invention to provide an isolated nucleicacid molecule which encodes a novel form of rhesus MC-5R, or rhesusMC-5R fragments, mutants or derivatives of SEQ ID NO:2. Any suchpolynucleotide includes but is not necessarily limited to nucleotidesubstitutions, deletions, additions, amino-terminal truncations andcarboxy-terminal truncations such that these mutations encode mRNA whichexpress a protein or protein fragment of diagnostic, therapeutic orprophylactic use and would be useful for screening for agonists and/orantagonists for vertebrate MC-5R function.

It is a further object of the present invention to provide therhesus-MC-5R proteins or protein fragments encoded by the nucleic acidmolecules referred to in the preceding paragraph.

It is a further object of the present invention to provide recombinantvectors and recombinant host cells which comprise a nucleic acidsequence encoding rhesus MC-5R or a biological equivalent thereof.

It is an object of the present invention to provide a substantiallypurified form of the rhesus MC-5R protein, as set forth in SEQ ID NO:2.

It is an object of the present invention to provide for biologicallyactive fragments and/or mutants of the rhesus MC-5R protein, such as setforth in SEQ ID NO:2, including but not necessarily limited to aminoacid substitutions, deletions, additions, amino terminal truncations andcarboxy-terminal truncations such that these mutations provide forproteins or protein fragments of diagnostic, therapeutic or prophylacticuse.

It is also an object of the present invention to provide for MC-5R-basedassays to select for modulators of this receptor protein. These assaysare preferably cell based assays whereby a DNA molecule encoding rhMC-5Ris transfected or transformed into a host cell, this recombinant hostcell is allowed to grow for a time sufficient to express MC-5R prior touse in various assays described herein.

It is a further object to provide for membrane preparations from hostcells transfected or transformed with a DNA molecule encoding rhMC-5Rfor use in assays to select for modulators of MC-5R activity.

It is also an object of the present invention to provide for MC-5R-basedin-frame fusion constructions, methods of expressing these fusionconstructs, biological equivalents disclosed herein, related assays,recombinant cells expressing these constructs, and agonistic and/orantagonistic compounds identified through the use of the nucleic acidencoding vertebrate MC-5R protein as well as the expressed protein.

As used herein, “rh” or refers to—rhesus—.

As used herein, “MC-5R” refers to—melanocortin 5 receptor—.

As used herein, “GPCR” refers to—G-protein coupled receptor—.

Whenever used herein, the term “mammalian host” will refer to anymammal, including a human being.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide sequence which encodes rhesus MC-5R, as setforth in SEQ ID NO:1.

FIG. 2A and FIG. 2B show the amino acid sequence of rhesus MC-5R as setforth in SEQ ID NO:2, as well as the open reading frame which encodesrhMC-5R, which is comprised within SEQ ID NO:1.

FIG. 3A and FIG. 3B show characterization of rhesus MC-5R. FIG. 3A showsinhibition of [¹²⁵I]NDP-α-MSH binding by NDP-α-MSH, γ2-MSH or Shu-9119.FIG. 3B shows stimulation of cAMP synthesis in response to NDP-α- MSH orγ2-MSH. Each curve represents a typical experiment with duplicatemeasurements. Average data from multiple experiments are listed in Table1.

FIG. 4 shows the comparison of binding affinity (IC50) and activationpotency (EC50) for the MC-5R from 2 species. Abbreviations are asfollows: A, human ACTH(1-24); α, α-MSH; γ, γ2-MSH; M, MT-II; N,NDP-α-MSH.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated nucleic acid molecule(polynucleotide) which encodes a novel rhesus monkey (Macaca mulatta)melanocortin-5 receptor (rhMC-5R). The nucleic acid molecules of thepresent invention are substantially free from other nucleic acids. Formost cloning purposes, DNA is a preferred nucleic acid.

The present invention relates to an isolated nucleic acid molecule(polynucleotide) which encodes mRNA which expresses a novel rhesusMC-5R, this DNA molecule comprising the nucleotide sequence disclosedherein as SEQ ID NO:1, shown herein as follows:

GCTGTCTTTC TTTGGTAACC TCTCTGGATT GTGAATTTAA AATGTGTTTT ACAGTAAATTTGCTGCCAAG ACAAGAGGTG AATTTCTCCA GCAATGAATT CCTCGTTTCA CCTGCATTTCTTGGATCTCA ACCTGAATGC CACAGAGGGC AACCTTTCAG GACCCAGTGT CAGAAACAAGTCTTCGCCCT GTGAAAACAT GGGCATGGCT GTGGAGGTGT TTCTCACTCT GGGTGCCATCAGCCTCGTGG AGAACATCTT GGTTATAGGG GCCATAGTGA AGAACAAAAA CCTGCACTGCCCCATGTACT TCTTCGTATG CAGCCTGGCG GTGGCCGACA TGCTGGTGAG CATGTCCAACGCCTGGGAGA CCATCACCAT CTACCTGCTC AACAACAAGC ACCTAGTGAT AGCAGACGCCTTTGTGCGCC ACATTGACAA CGTGTTTGAC TCCATGATCT GCATTTCCGT GGTGGCGTCCATGTGCAGCT TGCTGGCCAT TGCGGTGGAT AGGTACGTCA CCGTCTTCTA TGCCCTGCGCTACCACCACA TCATGACGGC GAGGCGCTCG GGGGCCATCA TCGCCGGCAT CTGGGCTTTCTGCACCGGGT GCGGCATCGT CTTCATCCTG TACTCCGAGT CCACCTACGT CATCCTGTGCCTCATCTCCA TGTTCTTCAC GATGCTCTTC CTCCTGGTGT GTCTGTACAT ACACATGTTCCTCCTGGCGC GGACTCACGC CAAGCGGATG GCGGCTCTGC CCGGGGCCAG CTCTGCGCGGCAGAGGACCA GCGTGCAGGG CGCGGTCACC CTCACCATGC TGCTGGGCGT GTTCATCGTGTGCTGGGCCC CGTTCTTCCT GCATCTCATT TTAATGCTTT CTTGCCCTCA GAACCTCTACTGCTCTTGCT TCATGTCTCA CTTCAATATG TACCTCATCC TCATCATGTG TAACTCCGTGGTGGACCCTC TCATATATGC CTTCCGCAGC CGGGAGATGC GCAAGACGTT TAAGGAGATTATTTGCTGCC GAGGCTTCCG AATCGCCTGC AGCTGTCCCG GAAGGGATTA AGTACAAAGT (SEQID NO:1).

The above-exemplified isolated DNA molecule, shown in FIG. 1 and setforth as SEQ ID NO:1, contains 1080 nucleotides. This DNA moleculecontains an open reading frame from nucleotide 94 to nucleotide 1068,with a “TAG” termination codon from nucleotides 1069-1071. This openreading frame encodes a rhesus MC-5R protein 325 amino acids in length,as shown in FIG. 2 and as set forth in SEQ ID NO:2.

The present invention also relates to biologically active fragments ormutants of SEQ ID NO:1 which encodes mRNA expressing a novel rhesusMC-5R. Any such biologically active fragment and/or mutant will encodeeither a protein or protein fragment which at least substantially mimicsthe pharmacological properties of a wild-type MC-5R protein, includingbut not limited to the rhesus MC-5R receptor protein as set forth in SEQID NO:2. Any such polynucleotide includes but is not necessarily limitedto nucleotide substitutions, deletions, additions, amino-terminaltruncations and carboxy-terminal truncations such that these mutationsencode mRNA which express a protein or protein fragment of diagnostic,therapeutic or prophylactic use and would be useful for screening foragonists and/or antagonists for MC-5R function.

A preferred aspect of this portion of the present invention is disclosedin FIG. 1, a rhesus cDNA molecule encoding a novel MC-5R (SEQ ID NO:1).

The isolated nucleic acid molecules of the present invention may includea deoxyribonucleic acid molecule (DNA), such as genomic DNA andcomplementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA).

It is known that there is a substantial amount of redundancy in thevarious codons which code for specific amino acids. Therefore, thisinvention is also directed to those DNA sequences encode RNA comprisingalternative codons which code for the eventual translation of theidentical amino acid, as shown below:

A=Ala=Alanine: codons GCA, GCC, GCG, GCU

C=Cys=Cysteine: codons UGC, UGU

D=Asp=Aspartic acid: codons GAC, GAU

E=Glu=Glutamic acid: codons GAA, GAG

F=Phe=Phenylalanine: codons UUC, UUU

G=Gly=Glycine: codons GGA, GGC, GGG, GGU

H=His =Histidine: codons CAC, CAU

I=Ile =Isoleucine: codons AUA, AUC, AUU

K=Lys=Lysine: codons AAA, AAG

L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU

M=Met=Methionine: codon AUG

N=Asp=Asparagine: codons AAC, AAU

P=Pro=Proline: codons CCA, CCC, CCG, CCU

Q=Gln=Glutamine: codons CAA, CAG

R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU

S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU

T=Thr-Threonine: codons ACA, ACC, ACG, ACU

V=Val=Valine: codons GUA, GUC, GUG, GUU

W=Trp=Tryptophan: codon UGG

Y=Tyr-Tyrosine: codons UAC, UAU

Therefore, the present invention discloses codon redundancy which mayresult in differing DNA molecules expressing an identical protein. Forpurposes of this specification, a sequence bearing one or more replacedcodons will be defined as a degenerate variation. Also included withinthe scope of this invention are mutations either in the DNA sequence orthe translated protein which do not substantially alter the ultimatephysical properties of the expressed protein. For example, substitutionof valine for leucine, arginine for lysine, or asparagine for glutaminemay not cause a change in functionality of the polypeptide.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide having properties that are different than those ofthe naturally occurring peptide. Methods of altering the DNA sequencesinclude but are not limited to site directed mutagenesis. Examples ofaltered properties include but are not limited to changes in theaffinity of an enzyme for a substrate or a receptor for a ligand.

Any of a variety of procedures may be used to clone rhesus MC-5R. Thesemethods include, but are not limited to, (1) a RACE PCR cloningtechnique (Frohman, et al., 1988, Proc. Natl. Acad. Sci. USA 85:8998-9002). 5′ and/or 3′ RACE may be performed to generate a full-lengthcDNA sequence. This strategy involves using gene-specificoligonucleotide primers for PCR amplification of rhesus MC-5R cDNA.These gene-specific primers are designed through identification of anexpressed sequence tag (EST) nucleotide sequence which has beenidentified by searching any number of publicly available nucleic acidand protein databases; (2) direct functional expression of the rhesusMC-5R cDNA following the construction of a rhesus MC-5R-containing cDNAlibrary in an appropriate expression vector system; (3) screening arhesus MC-5R-containing cDNA library constructed in a bacteriophage orplasmid shuttle vector with a labeled degenerate oligonucleotide probedesigned from the amino acid sequence of the rhesus MC-5R protein; (4)screening a rhesus MC-5R-containing cDNA library constructed in abacteriophage or plasmid shuttle vector with a partial cDNA encoding therhesus MC-5R protein. This partial cDNA is obtained by the specific PCRamplification of rhesus MC-5R DNA fragments through the design ofdegenerate oligonucleotide primers from the amino acid sequence knownfor other kinases which are related to the rhesus MC-5R protein; (5)screening a rhesus MC-5R-containing cDNA library constructed in abacteriophage or plasmid shuttle vector with a partial cDNA oroligonucleotide with homology to a mammalian MC-5R protein. Thisstrategy may also involve using gene-specific oligonucleotide primersfor PCR amplification of rhesus MC-5R cDNA identified as an EST asdescribed above; or (6) designing 5′ and 3′ gene specificoligonucleotides using SEQ ID NO: 1 as a template so that either thefull-length cDNA may be generated by known RACE techniques, or a portionof the coding region may be generated by these same known RACEtechniques to generate and isolate a portion of the coding region to useas a probe to screen one of numerous types of cDNA and/or genomiclibraries in order to isolate a fill-length version of the nucleotidesequence encoding rhesus MC-5R.

It is readily apparent to those skilled in the art that other types oflibraries, as well as libraries constructed from other cell types-orspecies types, may be useful for isolating a rhesus MC-5R- encoding DNAor a rhesus MC-5R homologue. Other types of libraries include, but arenot limited to, cDNA libraries derived from other rhesus cells.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have MC-5Ractivity. The selection of cells or cell lines for use in preparing acDNA library to isolate a cDNA encoding rhesus MC-5R may be done byfirst measuring cell-associated MC-5R activity using any known assayavailable for such a purpose.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Complementary DNA libraries may also be obtained from numerouscommercial sources, including but not limited to Clontech Laboratories,Inc. and Stratagene.

It is also readily apparent to those skilled in the art that DNAencoding rhesus MC-5R may also be isolated from a suitable genomic DNAlibrary. Construction of genomic DNA libraries can be performed bystandard techniques well known in the art. Well known genomic DNAlibrary construction techniques can be found in Sambrook, et al., supra.

In order to clone the rhesus MC-5R gene by one of the preferred methods,the amino acid sequence or DNA sequence of rhesus MC-5R or a homologousprotein may be necessary. To accomplish this, the MC-5R protein or ahomologous protein may be purified and partial amino acid sequencedetermined by automated sequenators. It is not necessary to determinethe entire amino acid sequence, but the linear sequence of two regionsof 6 to 8 amino acids can be determined for the PCR amplification of apartial rhesus MC-5R DNA fragment. Once suitable amino acid sequenceshave been identified, the DNA sequences capable of encoding them aresynthesized. Because the genetic code is degenerate, more than one codonmay be used to encode a particular amino acid, and therefore, the aminoacid sequence can be encoded by any of a set of similar DNAoligonucleotides. Only one member of the set will be identical to therhesus MC-5R sequence but others in the set will be capable ofhybridizing to rhesus MC-5R DNA even in the presence of DNAoligonucleotides with mismatches. The mismatched DNA oligonucleotidesmay still sufficiently hybridize to the rhesus MC-5R DNA to permitidentification and isolation of rhesus MC-5R encoding DNA.Alternatively, the nucleotide sequence of a region of an expressedsequence may be identified by searching one or more available genomicdatabases. Gene-specific primers may be used to perform PCRamplification of a cDNA of interest from either a cDNA library or apopulation of cDNAs. As noted above, the appropriate nucleotide sequencefor use in a PCR-based method may be obtained from SEQ ID NO:1, eitherfor the purpose of isolating overlapping 5′ and 3′ RACE products forgeneration of a full-length sequence coding for rhesus MC-5R, or toisolate a portion of the nucleotide sequence coding for rhesus MC-5R foruse as a probe to screen one or more cDNA-or genomic-based libraries toisolate a full-length sequence encoding rhesus MC-5R or rhesusMC-5R-like proteins.

Included in the present invention are DNA sequences that hybridize toSEQ ID NO:1 under stringent conditions. By way of example, and notlimitation, a procedure using conditions of high stringency is asfollows: Prehybridization of filters containing DNA is carried out for 2hours to overnight at 65° C. in buffer composed of 6×SSC, 5×Denhardt'ssolution, and 100 μg/ml denatured salmon sperm DNA. Filters arehybridized for 12 to 48 hrs at 65° C. in prehybridization mixturecontaining 100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of³²P-labeled probe. Washing of filters is done at 37° C. for 1 hr in asolution containing 2×SSC, 0.1% SDS. This is followed by a wash in0.1×SSC, 0.1% SDS at 50° C. for 45 min. before autoradiography. Otherprocedures using conditions of high stringency would include either ahybridization step carried out in 5×SSC, 5×Denhardt's solution, 50%formamide at 42° C. for 12 to 48 hours or a washing step carried out in0.2×SSPE, 0.2% SDS at 65° C. for 30 to 60 minutes.

Reagents mentioned in the foregoing procedures for carrying out highstringency hybridization are well known in the art. Details of thecomposition of these reagents can be found in, e.g., Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. In addition to the foregoing, otherconditions of high stringency which may be used are well known in theart.

Melanocortin receptors belong to the rhodopsin sub-family of GPCR's.However, several features in the rhMC-5R are shared with all otherreceptors and are absent in most other GPCR's, including the EN motif inTM1, the lack of Cys in the loop between TM2 and TM3 or between TM4 andTM5, the MxxxxxxxY motif in TM5, and the DPxxY motif in TM7. Since allmelanocortin receptors lack Cys residues in the extracellular loops thatare present in other members of the rhodopsin sub-family, interhelicaldisulfide bond (e.g., between the Cys residues near the top of TM3 andTM5) may play the same function as interloop disulfide bond in mostother GPCR's.

The present invention also relates to a substantially purified form ofthe rhesus melanocortin-5 receptor protein, which comprises the aminoacid sequence is disclosed in FIG. 2 and set forth as SEQ ID NO:2.

The present invention also relates to biologically active fragmentsand/or mutants of the rhesus melanocortin-5 receptor protein comprisingthe amino acid sequence set forth as SEQ ID NO:2, including but notnecessarily limited to amino acid substitutions, deletions, additions,amino terminal truncations and carboxy-terminal truncations such thatthese mutations provide for proteins or protein fragments of diagnostic,therapeutic or prophylactic use and would be useful for screening foragonists and/or antagonists for MC-5R function.

A preferred aspect of the present invention is disclosed in FIG. 2 andis set forth as SEQ ID NO:2, and as herein set forth as follows:

M N S S F H L H F L D L N L N A T E G N L S G P S V R N K S S P C E N MG M A V E V F L T L G A I S L V E N I L V I G A I V K N K N L H C P M YF F V C S L A V A D M L V S M S N A W E T I T I Y L L N N K H L V I A DA F V R H I D N V F D S M I C I S V V A S M C S L L A I A V D R Y V T VF Y A L R Y H H I M T A R R S G A I I A G I W A F C T G C G I V F I L YS E S T Y V I L C L I S M F F T M L F L L V C L Y I H M F L L A R T H AK R M A A L P G A S S A R Q R T S V Q G A V T L T M L L G V F I V C W AP F F L H L I L M L S C P Q N L Y C S C F M S H F N M Y L I L I M C N SV V D P L I Y A F R S R E M R K T F K E I I C C R G F R I A C S C P G RD (SEQ ID NO:2)

which comprises the amino acid sequence of wild type rhesusmelanocortin-5 recptor protein.

As with many receptor proteins, it is possible to modify many of theamino acids, particularly those which are not found in the ligandbinding domain, and still retain substantially the same biologicalactivity as the original receptor. Thus this invention includes modifiedrhMC-5R polypeptides which have amino acid deletions, additions, orsubstitutions but that still retain substantially the same biologicalactivity as rhMC-5R. It is generally accepted that single amino acidsubstitutions do not usually alter the biological activity of a protein(see, e.g., Molecular Biology of the Gene, Watson et al., 1987, FourthEd., The Benjamin/Cummings Publishing Co., Inc., page 226; andCunningham & Wells, 1989, Science 244:1081-1085). Accordingly, thepresent invention includes isolated nucleic acid molecules and expressedMC-5R proteins wherein one amino acid substitution is generated andwhich this protein retains substantially the same biological activity aswild-type rhMC-5R. The present invention also includes isolated nucleicacid molecules and expressed MC-5R proteins wherein two or more aminoacid substitution is generated wherein this protein retainssubstantially the same biological activity as wild-type rhMC-5R. Inparticular, the present invention includes embodiments where theabove-described substitutions are conservative substitutions. Inparticular, the present invention includes embodiments where theabove-described substitutions do not occur in the ligand-binding domainof rhMC-5R.

Following expression of MC-5R in a host cell, MC-5R protein may berecovered to provide MC-5R protein in active form. Several MC-5R proteinpurification procedures are available and suitable for use. RecombinantMC-5R protein may be purified from cell lysates and extracts by variouscombinations of, or individual application of salt fractionation, ionexchange chromatography, size exclusion chromatography, hydroxylapatiteadsorption chromatography and hydrophobic interaction chromatography. Inaddition, recombinant MC-5R protein can be separated from other cellularproteins by use of an immunoaffinity column made with monoclonal orpolyclonal antibodies specific for full-length MC-5R protein, orpolypeptide fragments of MC-5R protein.

The present invention also relates to isolated nucleic acid moleculeswhich are fusion constructions expressing fusion proteins useful inassays to identify compounds which modulate wild-type vertebrate MC-5Ractivity. A preferred aspect of this portion of the invention includes,but is not limited to, glutathione S-transferase (GST)-MC-5R fusionconstructs which include, but are not limited to, either theintracellular domain of rhesus MC-5R as an in-frame fusion at thecarboxy terminus of the GST gene or the extracellular and transmembraneligand binding domain of MC5R fused to an GST or immunoglobulin gene bymethods known to one of ordinary skill in the art. Recombinant GST-MC-5Rfusion proteins may be expressed in various expression systems,including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using abaculovirus expression vector (pAcG2T, Pharmingen).

The present invention also relates to subcellular membrane fractionsfrom the recombinant host cells (both prokaryotic and eukaryotic as wellas both stably and transiently transformed cells) which contain thenucleic acids of the present invention. These subcellular membranefractions will comprise either wild-type or mutant forms of rhesusmelanocortin-5 receptor proteins at levels substantially aboveendogenous levels and hence will be useful in various assays describedthroughout this specification.

The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, which contain thesubstantially purified nucleic acid molecules disclosed throughout thisspecification. The nucleic acid molecules of the present inventionencoding rhMC-5R, in whole or in part, can be linked with other DNAmolecules, i.e., DNA molecules to which the rhMC-5R are not naturallylinked, to form “recombinant DNA molecules” containing the receptor. Thenovel DNA sequences of the present invention can be inserted intovectors which comprise nucleic acids encoding a rhMC-5R or a functionalequivalent. These vectors may be comprised of DNA or RNA; for mostcloning purposes DNA vectors are preferred. Typical vectors includeplasmids, modified viruses, bacteriophage and cosmids, yeast artificialchromosomes and other forms of episomal or integrated DNA that canencode a rhMC-5R. It is well within the skilled artisan to determine anappropriate vector for a particular gene transfer or other use.

To this end, the present invention also includes vectors containing anrhMC-5R gene, host cells containing the vectors, and methods of makingsubstantially pure rhMC-5R protein comprising the steps of introducingthe rhMC-5R gene into a host cell, and cultivating the host cell underappropriate conditions such that rhMC-5R is produced. The rhMC-5R soproduced may be harvested from the host cells in conventional ways.Therefore, the present invention also relates to methods of expressingthe rhesus MC-5R protein and biological equivalents disclosed herein,assays employing these gene products, recombinant host cells whichcomprise DNA constructs which express these receptor proteins, andcompounds identified through these assays which act as agonists orantagonists of MC-5R activity.

The cloned rhesus MC-5R cDNA obtained through the methods describedabove may be recombinantly expressed by molecular cloning into anexpression vector (such as pcDNA3.neo, pcDNA3.1, pCR2.1, pBlueBacHis2 orpLITMUS28) containing a suitable promoter and other appropriatetranscription regulatory elements, and transferred into prokaryotic oreukaryotic host cells to produce recombinant rhesus MC-5R. Techniquesfor such manipulations can be found described in Sambrook, et al.,supra, are discussed at length in the Example section and are well knownand easily available to the artisan of ordinary skill in the art.

A variety of mammalian expression vectors may be used to expressrecombinant rhesus MC-5R in mammalian cells. Expression vectors aredefined herein as DNA sequences that are required for the transcriptionof cloned DNA and the translation of their mRNAs in an appropriate host.Such vectors can be used to express eukaryotic DNA in a variety of hostssuch as bacteria, blue green algae, plant cells, insect cells and animalcells. Specifically designed vectors allow the shuttling of DNA betweenhosts such as bacteria-yeast or bacteria-animal cells. An appropriatelyconstructed expression vector should contain: an origin of replicationfor autonomous replication in host cells, selectable markers, a limitednumber of useful restriction enzyme sites, a potential for high copynumber, and active promoters. A promoter is defined as a DNA sequencethat directs RNA polymerase to bind to DNA and initiate RNA synthesis. Astrong promoter is one which causes mRNAs to be initiated at highfrequency. Expression vectors may include, but are not limited to,cloning vectors, modified cloning vectors, specifically designedplasmids or viruses. Commercially available mammalian expression vectorswhich may be suitable for recombinant rhesus MC-5R expression, includebut are not limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen),pCI-neo (Promega), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (NewEngland Bioloabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen),pMClneo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo(ATCC 37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC37146), pUCTag (ATCC 37460), and γZD35 (ATCC 37565).

Also, a variety of bacterial expression vectors may be used to expressrecombinant rhesus MC-5R in bacterial cells. Commercially availablebacterial expression vectors which may be suitable for recombinantrhesus MC-5R expression include, but are not limited to pCR2.1(Invitrogen), pET11a (Novagen), lambda gt11a (Invitrogen), and pKK223-3(Pharmacia).

In addition, a variety of fungal cell expression vectors may be used toexpress recombinant rhesus MC-5R in fungal cells. Commercially availablefungal cell expression vectors which may be suitable for recombinantrhesus MC-5R expression include but are not limited to pYES2(Invitrogen) and Pichia expression vector (Invitrogen).

Also, a variety of insect cell expression vectors may be used to expressrecombinant receptor in insect cells. Commercially available insect cellexpression vectors which may be suitable for recombinant expression ofrhesus MC-5R include but are not limited to pBlueBacIII and pBlueBacHis2(Invitrogen), and pAcG2T (Pharmingen).

Expression of rhesus MC-5R DNA may also be performed using in vitroproduced synthetic mRNA. Synthetic mRNA can be efficiently translated invarious cell-free systems, including but not limited to wheat germextracts and reticulocyte extracts, as well as efficiently translated incell based systems, including but not limited to microinjection intofrog oocytes, with microinjection into frog oocytes being preferred.

To determine the rhesus MC-5R cDNA sequence(s) that yields optimallevels of rhesus MC-5R, cDNA molecules including but not limited to thefollowing can be constructed: a cDNA fragment containing the full-lengthopen reading frame for rhesus MC-5R as well as various constructscontaining portions of the cDNA encoding only specific domains of theprotein or rearranged domains of the protein. All constructs can bedesigned to contain none, all or portions of the 5′ and/or 3′untranslated region of a rhesus MC-5R cDNA. The expression levels andactivity of rhesus MC-5R can be determined following the introduction,both singly and in combination, of these constructs into appropriatehost cells. Following determination of the rhesus MC-5R cDNA cassetteyielding optimal expression in transient assays, this MC-5R cDNAconstruct is transferred to a variety of expression vectors (includingrecombinant viruses), including but not limited to those for mammaliancells, plant cells, insect cells, oocytes, bacteria, and yeast cells.

Therefore, another aspect of the present invention includes host cellsthat have been engineered to contain and/or express DNA sequencesencoding the rhMC-5R. Such recombinant host cells can be cultured undersuitable conditions to produce rhMC-5R or a biologically equivalentform. Recombinant host cells may be prokaryotic or eukaryotic, includingbut not limited to, bacteria such as E. coli, fingal cells such asyeast, mammalian cells including, but not limited to, cell lines ofhuman, bovine, porcine, monkey and rodent origin, and insect cellsincluding but not limited to Drosophila and silkworm derived cell lines.Therefore, an expression vector containing DNA encoding a rhesusMC-5R-like protein may be used for expression of rhesus MC-5R in arecombinant host cell. Recombinant host cells may be prokaryotic oreukaryotic, including but not limited to bacteria such as E. coli,fungal cells such as yeast, mammalian cells including but not limited tocell lines of human, bovine, porcine, monkey and rodent origin, andinsect cells including but not limited to Drosophila andsilkworm-derived cell lines. For instance, one insect expression systemutilizes Spodoptera frugiperda (Sf21) insect cells (Invitrogen) intandem with a baculovirus expression vector (pAcG2T, Pharmingen). Also,mammalian species which may be suitable and which are commerciallyavailable, include but are not limited to, L cells L-M(TK⁻) (ATCC CCL1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650),COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3(ATCC CRL 1658), HeLa (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCCCCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL 209). The expressionvector may be introduced into host cells via any one of a number oftechniques including but not limited to transformation, transfection,protoplast fusion, and electroporation. The expression vector-containingcells are individually analyzed to determine whether they produce rhesusMC-5R protein. Identification of rhesus MC-5R expressing cells may bedone by several means, including but not limited to immunologicalreactivity with anti-rhesus MC-5R antibodies, labeled ligand binding andthe presence of host cell-associated rhesus MC-5R activity.

The assays described herein as well as protein purification schemes canbe carried out with cells that have been transiently or stablytransfected or transformed with an expression vector which directsexpression of MC-5R. The expression vector may be introduced into hostcells via any one of a number of techniques including but not limited totransformation, transfection, protoplast fusion, and electroporation.Transformation is meant to encompass a genetic change to the target cellresulting from an incorporation of DNA. Transfection is meant to includeany method known in the art for introducing MC-5R into the test cells.For example, transfection includes calcium phosphate or calcium chloridemediated transfection, lipofection, infection with a retroviralconstruct containing MC-5R, and electroporation. The expressionvector-containing cells are individually analyzed to determine whetherthey produce human MC-5R protein. Identification of human MC-5Rexpressing cells may be done by several means, including but not limitedto immunological reactivity with anti-human MC-5R antibodies, labeledligand binding and the presence of host cell-associated human MC-5Ractivity.

The specificity of binding of compounds showing affinity for rhMC-5R isshown by measuring the affinity of the compounds for recombinant cellsexpressing the cloned receptor or for membranes from these cells.Expression of the cloned receptor and screening for compounds that bindto rhMC-5R or that inhibit the binding of a known, radiolabeled ligandof rhMC-5R to these cells, or membranes prepared from these cells,provides an effective method for the rapid selection of compounds withhigh affinity for rhMC-5R. Such ligands need not necessarily beradiolabeled but can also be nonisotopic compounds that can be used todisplace bound radiolabeled compounds or that can be used as activatorsin functional assays. Compounds identified by the above method arelikely to be agonists or antagonists of rhMC-5R and may be peptides,proteins, or non-proteinaceous organic molecules.

Accordingly, the present invention is directed to methods for screeningfor compounds which modulate the expression of DNA or RNA encoding aMC-5R protein as well as compounds which effect the function of theMC-5R protein. Methods for identifying agonists and antagonists of otherreceptors are well known in the art and can be adapted to identifyagonists and antagonists of MC-5R. For example, Cascieri et al. (1992,Molec. Pharmacol. 41:1096-1099) describe a method for identifyingsubstances that inhibit agonist binding to rat neurokinin receptors andthus are potential agonists or antagonists of neurokinin receptors. Themethod involves transfecting COS cells with expression vectorscontaining rat neurokinin receptors, allowing the transfected cells togrow for a time sufficient to allow the neurokinin receptors to beexpressed, harvesting the transfected cells and resuspending the cellsin assay buffer containing a known radioactively labeled agonist of theneurokinin receptors either in the presence or the absence of thesubstance, and then measuring the binding of the radioactively labeledknown agonist of the neurokinin receptor to the neurokinin receptor. Ifthe amount of binding of the known agonist is less in the presence ofthe substance than in the absence of the substance, then the substanceis a potential agonist or antagonist of the neurokinin receptor. Wherebinding of the substance such as an agonist or antagonist to MC-5R ismeasured, such binding can be measured by employing a labeled substanceor agonist. The substance or agonist can be labeled in any convenientmanner known to the art, e.g., radioactively, fluorescently,enzymatically.

Therefore, the specificity of binding of compounds having affinity forMC-5R is shown by measuring the affinity of the compounds forrecombinant cells expressing the cloned receptor or for membranes fromthese cells. Expression of the cloned receptor and screening forcompounds that bind to MC-5R or that inhibit the binding of a known,radiolabeled ligand of MC-5R to these cells, or membranes prepared fromthese cells, provides an effective method for the rapid selection ofcompounds with high affinity for MC-5R. Such ligands need notnecessarily be radiolabeled but can also be nonisotopic compounds thatcan be used to displace bound radiolabeled compounds or that can be usedas activators in functional assays. Compounds identified by the abovemethod are likely to be agonists or antagonists of MC-5R and may bepeptides, proteins, or non-proteinaceous organic molecules. Compoundsmay modulate by increasing or attenuating the expression of DNA or RNAencoding MC-5R, or by acting as an agonist or antagonist of the MC-5Rreceptor protein. These compounds that modulate the expression of DNA orRNA encoding MC-5R or the biological function thereof may be detected bya variety of assays. The assay may be a simple “yes/no” assay todetermine whether there is a change in expression or function. The assaymay be made quantitative by comparing the expression or function of atest sample with the levels of expression or function in a standardsample. Kits containing MC-5R, antibodies to MC-5R, or modified MC-5Rmay be prepared by known methods for such uses.

Therefore, the present invention relates to methods of expressingrhMC-5R in recombinant systems and of identifying agonists andantagonists of rhMC-5R. When screening compounds in order to identifypotential pharmaceuticals that specifically interact with a targetreceptor, it is necessary to ensure that the compounds identified are asspecific as possible for the target receptor. To do this, it isnecessary to screen the compounds against as wide an array as possibleof receptors that are similar to the target receptor. Thus, in order tofind compounds that are potential pharmaceuticals that interact withreceptor A, it is necessary not only to ensure that the compoundsinteract with receptor A (the “plus target”) and produce the desiredpharmacological effect through receptor A, it is also necessary todetermine that the compounds do not interact with receptors B, C, D,etc. (the “minus targets”). In general, as part of a screening program,it is important to have as many minus targets as possible (see Hodgson,1992, Bio/Technology 10:973-980). Rhesus MC-5R proteins and the DNAmolecules encoding this receptor protein have the additional utility inthat they can be used as “minus targets” in screens designed to identifycompounds that specifically interact with other G-protein coupledreceptors. Due to homology to GPCRs, the rhMC-5R of this invention isbelieved to function similarly to GPCRs and have similar biologicalactivity. They are useful in understanding the biological andphysiological effects in the rhesus to in identify melanocortin activeprocess in primates, followed by human clinical trials. More notable,rhMC-5R agonists will be identified and evaluated for their effects onfood intake, weight gain, and metabolic rate to identifynovel-anti-obesity agents that are effective in primates. They may alsobe used to scan for rhesus monkey melanocortin agonists and antagonists;as in particular to test the specificity of identified ligands.

To this end, the present invention relates in part to methods ofidentifying a substance which modulates MC-5R receptor activity, whichinvolves:

(a) combining a test substance in the presence and absence of a MC-5Rreceptor protein wherein said MC-5R receptor protein comprises the aminoacid sequence as set forth in SEQ ID NO:2; and,

(b) measuring and comparing the effect of the test substance in thepresence and absence of the MC-5R receptor protein.

In addition, several specific embodiments are disclosed herein to showthe diverse type of screening or selection assay which the skilledartisan may utilize in tandem with an expression vector directing theexpression of the MC-5R receptor protein. Methods for identifyingagonists and antagonists of other receptors are well known in the artand can be adapted to identify agonists and antagonists of MC-5R.Therefore, these embodiments are presented as examples and not aslimitations. To this end, the present invention includes assays by whichMC-5R modulators (such as agonists and antagonists) may be identified.Accordingly, the present invention includes a method for determiningwhether a substance is a potential agonist or antagonist of MC-5R thatcomprises:

(a) transfecting or transforming cells with an expression vector thatdirects expression of MC-5R in the cells, resulting in test cells;

(b) allowing the test cells to grow for a time sufficient to allow MC-5Rto be expressed;

(c) exposing the cells to a labeled ligand of MC-5R in the presence andin the absence of the substance;

(d) measuring the binding of the labeled ligand to MC-5R; where if theamount of binding of the labeled ligand is less in the presence of thesubstance than in the absence of the substance, then the substance is apotential agonist or antagonist of MC-5R.

The conditions under which step (c) of the method is practiced areconditions that are typically used in the art for the study ofprotein-ligand interactions: e.g., physiological pH; salt conditionssuch as those represented by such commonly used buffers as PBS or intissue culture media; a temperature of about 4° C. to about 55° C. Thetest cells may be harvested and resuspended in the presence of thesubstance and the labeled ligand. In a modification of theabove-described method, step (c) is modified in that the cells are notharvested and resuspended but rather the radioactively labeled knownagonist and the substance are contacted with the cells while the cellsare attached to a substratum, e.g., tissue culture plates.

The present invention also includes a method for determining whether asubstance is capable of binding to MC-5R, i.e., whether the substance isa potential agonist or an antagonist of MC-5R, where the methodcomprises:

(a) transfecting or transforming cells with an expression vector thatdirects the expression of MC-5R in the cells, resulting in test cells;

(b) exposing the test cells to the substance;

(c) measuring the amount of binding of the substance to MC-5R;

(d) comparing the amount of binding of the substance to MC-5R in thetest cells with the amount of binding of the substance to control cellsthat have not been transfected with MC-5R;

wherein if the amount of binding of the substance is greater in the testcells as compared to the control cells, the substance is capable ofbinding to MC-5R. Determining whether the substance is actually anagonist or antagonist can then be accomplished by the use of functionalassays such as, e.g., the assay involving the use of promiscuousG-proteins described below.

The conditions under which step (b) of the method is practiced areconditions that are typically used in the art for the study ofprotein-ligand interactions: e.g., physiological pH; salt conditionssuch as those represented by such commonly used buffers as PBS or intissue culture media; a temperature of about 4° C. to about 55° C. Thetest cells are harvested and resuspended in the presence of thesubstance.

Chen et al. (1995, Analytical Biochemistry 226: 349-354) describe acolorometric assay which utilizes a recombinant cell transfected with anexpression vector encoding a G-protein coupled receptor with a secondexpression vector containing a promoter with a cAMP responsive elementfused to the LacZ gene. Activity of the overexpressed G-protein coupledreceptor is measured as the expression and OD measurement of β-Gal.Therefore, another aspect of this portion of the invention includes anon-radioactive method for determining whether a substance is apotential agonist or antagonist of MC-5R that comprises:

(a) transfecting or transforming cells with an expression vectorencoding MC-5R, resulting in test cells;

(b) transfecting or transforming the test cells of step (a) with anexpression vector which comprises a cAMP-inducible promoter fused to acolorometric gene such a LacZ;

(c) allowing the transfected cells to grow for a time sufficient toallow MC-5R to be expressed;

(d) harvesting the transfected cells and resuspending the cells in thepresence of a known agonist of MC-5R and/or in both the presence andabsence of the test compound;

(e) measuring the binding of the known agonist and test compound tooverexpressed MC-5R by a colorometric assay which measures expressionoff the cAMP-inducible promoter and comparing expression levels in thepresence of the known agonist as well as in the presence and absence ofthe unknown substance so as to determine whether the unknown substanceacts as either a potential agonist or antagonist of MC-5R.

Additional methods of identifying agonists or antagonists include butare by no means limited to the following:

I. (a) transfecting or transforming cells with a first expression vectorwhich directs expression of MC-5R and a second expression vector whichdirects the expression of a promiscuous G-protein, resulting in testcells;

(b) exposing the test cells to a substance that is a suspected agonistof MC-5R;

(c) measuring the level of inositol phosphates in the cells; where anincrease in the level of inositol phosphates in the cells as compared tothe level of inositol phosphates in the cells in the absence of thesuspected agonist indicates that the substance is an agonist of MC-5R.

II. (a) transfecting or transforming cells with a first expressionvector which directs expression of MC-5R and a second expression vectorwhich directs the expression of a promiscuous G-protein, resulting intest cells;

(b) exposing the test cells to a substance that is an agonist of MC-5R;

(c) subsequently or concurrently to step (b), exposing the test cells toa substance that is a suspected antagonist of MC-5R;

(d) measuring the level of inositol phosphates in the cells;

where a decrease in the level of inositol phosphates in the cells in thepresence of the suspected antagonist as compared to the level ofinositol phosphates in the cells in the absence of the suspectedantagonist indicates that the substance is an antagonist of MC-5R.

III. the method of II wherein the first and second expression vectors ofstep (a) are replaced with a single expression vector which expresses achimeric MC-5R protein fused at its C-terminus to a promiscuousG-protein.

The above-described methods can be modified in that, rather thanexposing the test cells to the substance, membranes can be prepared fromthe test cells and those membranes can be exposed to the substance. Sucha modification utilizing membranes rather than cells is well known inthe art and is described in, e.g., Hess et al., 1992, Biochem. Biophys.Res. Comm. 184:260-268. Accordingly, another embodiment of the presentinvention includes a method for determining whether a substance bindsand/or is a potential agonist or antagonist of MC-5R wherein membranepreparations from the test cells are utilized in place of the testcells. Such methods comprise the following and may utilized thephysiological conditions as noted above:

(a) transfecting or transforming cells with an expression vector thatdirects the expression of MC-5R in the cells, resulting in test cells;

(b) preparing membranes containing MC-5R from the test cells andexposing the membranes to a ligand of MC-5R under conditions such thatthe ligand binds to the MC-5R in the membranes;

(c) subsequently or concurrently to step (b), exposing the membranesfrom the test cells to a substance;

(d) measuring the amount of binding of the ligand to the MC-5R in themembranes in the presence and the absence of the substance;

(e) comparing the amount of binding of the ligand to MC-5R in themembranes in the presence and the absence of the substance where adecrease in the amount of binding of the ligand to MC-5R in themembranes in the presence of the substance indicates that the substanceis capable of binding to MC-5R.

The present invention also relates to a method for determining whether asubstance is capable of binding to MC-5R comprising:

(a) transfecting or transforming cells with an expression vector thatdirects the expression of MC-5R in the cells, resulting in test cells;

(b) preparing membranes containing MC-5R from the test cells andexposing the membranes from the test cells to the substance;

(c) measuring the amount of binding of the substance to the MC-5R in themembranes from the test cells;

(d) comparing the amount of binding of the substance to MC-5R in themembranes from the test cells with the amount of binding of thesubstance to membranes from control cells that have not been transfectedwith MC-5R, where if the amount of binding of the substance to MC-5R inthe membranes from the test cells is greater than the amount of bindingof the substance to the membranes from the control cells, then thesubstance is capable of binding to MC-5R.

A preferred embodiment of the present invention is determining variousligand binding affinities using ¹²⁵I-labeled NDP-α-MSH as the labeledligand in the presence of varying concentration of unlabeled ligands.The activation of the second messenger pathway may be determined bymeasuring the intracellular cAMP elicited by agonist at variousconcentration.

The present invention also relates to polyclonal and monoclonalantibodies raised in response to either the rhesus form of MC-5R, or abiologically active fragment thereof. Polyclonal or monoclonalantibodies may be raised against rhesus MC-5R or a synthetic peptide(usually from about 9 to about 25 amino acids in length) from a portionof rhesus MC-5R as disclosed in SEQ ID NO:2. Monospecific antibodies torhesus MC-5R are purified from mammalian antisera containing antibodiesreactive against rhesus MC-5R or are prepared as monoclonal antibodiesreactive with rhesus MC-5R using the technique of Kohler and Milstein(1975, Nature 256: 495-497). Monospecific antibody as used herein isdefined as a single antibody species or multiple antibody species withhomogenous binding characteristics for rhesus MC-5R. Homogenous bindingas used herein refers to the ability of the antibody species to bind toa specific antigen or epitope, such as those associated with rhesusMC-5R, as described above. Rhesus MC-5R-specific antibodies are raisedby immunizing animals such as mice, rats, guinea pigs, rabbits, goats,horses and the like, with an appropriate concentration of rhesus MC-5Rprotein or a synthetic peptide generated from a portion of rhesus MC-5Rwith or without an immune adjuvant.

Preimmune serum is collected prior to the first immunization. Eachanimal receives between about 0.1 μg and about 1000 μg of rhesus MC-5Rprotein associated with an acceptable immune adjuvant. Such acceptableadjuvants include, but are not limited to, Freund's complete, Freund'sincomplete, alum-precipitate, water in oil emulsion containingCorynebacterium parvum and tRNA. The initial immunization consists ofrhesus MC-5R protein or peptide fragment thereof in, preferably,Freund's complete adjuvant at multiple sites either subcutaneously (SC),intraperitoneally (IP) or both. Each animal is bled at regularintervals, preferably weekly, to determine antibody titer. The animalsmay or may not receive booster injections following the initialimmunization. Those animals receiving booster injections are generallygiven an equal amount of rhesus MC-5R in Freund's incomplete adjuvant bythe same route. Booster injections are given at about three weekintervals until maximal titers are obtained. At about 7 days after eachbooster immunization or about weekly after a single immunization, theanimals are bled, the serum collected, and aliquots are stored at about−20° C.

Monoclonal antibodies (mAb) reactive with rhesus MC-5R are prepared byimmunizing inbred mice, preferably Balb/c, with rhesus MC-5R protein.The mice are immunized by the IP or SC route with about 1 μg to about100 μg, preferably about 10 μg, of rhesus MC-5R protein in about 0.5 mlbuffer or saline incorporated in an equal volume of an acceptableadjuvant, as discussed above. Freund's complete adjuvant is preferred.The mice receive an initial immunization on day 0 and are rested forabout 3 to about 30 weeks. Immunized mice are given one or more boosterimmunizations of about 1 to about 100 μg of rhesus MC-5R in a buffersolution such as phosphate buffered saline by the intravenous (IV)route. Lymphocytes, from antibody positive mice, preferably spleniclymphocytes, are obtained by removing spleens from immunized mice bystandard procedures known in the art. Hybridoma cells are produced bymixing the splenic lymphocytes with an appropriate fusion partner,preferably myeloma cells, under conditions which will allow theformation of stable hybridomas. Fusion partners may include, but are notlimited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp 2/0, withSp 2/0 being preferred. The antibody producing cells and myeloma cellsare fused in polyethylene glycol, about 1000 mol. wt., at concentrationsfrom about 30% to about 50%. Fused hybridoma cells are selected bygrowth in hypoxanthine, thymidine and aminopterin supplementedDulbecco's Modified Eagles Medium (DMEM) by 25 procedures known in theart. Supernatant fluids are collected from growth positive wells onabout days 14, 18, and 21 and are screened for antibody production by animmunoassay such as solid phase immunoradioassay (SPIRA) using rhesusMC-5R as the antigen. The culture fluids are also tested in theOuchterlony precipitation assay to determine the isotype of the mAb.Hybridoma cells from antibody positive wells are cloned by a techniquesuch as the soft agar technique of MacPherson, 1973, Soft AgarTechniques, in Tissue Culture Methods and Applications, Kruse andPaterson, Eds., Academic Press.

Monoclonal antibodies are produced in vivo by injection of pristineprimed Balb/c mice, approximately 0.5 ml per mouse, with about 2×10⁶ toabout 6×10⁶ hybridoma cells about 4 days after priming. Ascites fluid iscollected at approximately 8-12 days after cell transfer and themonoclonal antibodies are purified by techniques known in the art.

In vitro production of anti-rhesus MC-5R mAb is carried out by growingthe hybridoma in DMEM containing about 2% fetal calf serum to obtainsufficient quantities of the specific mAb. The mAb are purified bytechniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of rhesusMC-5R in body fluids or tissue and cell extracts.

It is readily apparent to those skilled in the art that the abovedescribed methods for producing monospecific antibodies may be utilizedto produce antibodies specific for rhesus MC-5R peptide fragments, orfull-length rhesus MC-5R.

Rhesus MC-5R antibody affinity columns are made, for example, by addingthe antibodies to Affigel-10 (Biorad), a gel support which ispre-activated with N-hydroxysuccinimide esters such that the antibodiesform covalent linkages with the agarose gel bead support. The antibodiesare then coupled to the gel via amide bonds with the spacer arm. Theremaining activated esters are then quenched with 1M ethanolamine HCl(pH 8). The column is washed with water followed by 0.23 M glycine HCl(pH 2.6) to remove any non-conjugated antibody or extraneous protein.The column is then equilibrated in phosphate buffered saline (pH 7.3)and the cell culture supernatants or cell extracts containingfull-length rhesus MC-5R or rhesus MC-5R protein fragments are slowlypassed through the column. The column is then washed with phosphatebuffered saline until the optical density (A₂₈₀) falls to background,then the protein is eluted with 0.23 M glycine-HCl (pH 2.6). Thepurified rhesus MC-5R protein is then dialyzed against phosphatebuffered saline.

The DNA molecules, RNA molecules, recombinant protein and antibodies ofthe present invention may be used to screen and measure levels of rhesusMC-5R. The recombinant proteins, DNA molecules, RNA molecules andantibodies lend themselves to the formulation of kits suitable for thedetection and typing of rhesus MC-5R. Such a kit would comprise acompartmentalized carrier suitable to hold in close confinement at leastone container. The carrier would further comprise reagents such asrecombinant MC-5R or anti-MC-5R antibodies suitable for detecting rhesusMC-5R. The carrier may also contain a means for detection such aslabeled antigen or enzyme substrates or the like.

Pharmaceutically useful compositions comprising modulators of rhesusMC-5R may be formulated according to known methods such as by theadmixture of a pharmaceutically acceptable carrier. Examples of suchcarriers and methods of formulation may be found in Remington'sPharmaceutical Sciences. To form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the protein, DNA, RNA, modifiedrhesus MC-5R, or either MC-5R agonsits or antagonists including tyrosinekinase activators or inhibitors.

Therapeutic or diagnostic compositions of the invention are administeredto an individual in amounts sufficient to treat or diagnose disorders.The effective amount may vary according to a variety of factors such asthe individual's condition, weight, sex and age. Other factors includethe mode of administration.

The pharmaceutical compositions may be provided to the individual by avariety of routes such as subcutaneous, topical, oral and intramuscular.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties which are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein may beused alone at appropriate dosages. Alternatively, co-administration orsequential administration of other agents may be desirable.

The present invention also has the objective of providing suitabletopical, oral, systemic and parenteral pharmaceutical formulations foruse in the novel methods of treatment of the present invention. Thecompositions containing compounds identified according to this inventionas the active ingredient can be administered in a wide variety oftherapeutic dosage forms in conventional vehicles for administration.For example, the compounds can be administered in such oral dosage formsas tablets, capsules (each including timed release and sustained releaseformulations), pills, powders, granules, elixirs, tinctures, solutions,suspensions, syrups and emulsions, or by injection. Likewise, they mayalso be administered in intravenous (both bolus and infusion),intraperitoneal, subcutaneous, topical with or without occlusion, orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts.

Advantageously, compounds of the present invention may be administeredin a single daily dose, or the total daily dosage may be administered individed doses of two, three or four times daily. Furthermore, compoundsfor the present invention can be administered in intranasal form viatopical use of suitable intranasal vehicles, or via transdermal routes,using those forms of transdermal skin patches well known to those ofordinary skill in that art. To be administered in the form of atransdermal delivery system, the dosage administration will, of course,be continuous rather than intermittent throughout the dosage regimen.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing the compounds of the present invention isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal, hepatic and cardiovascular function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

The following examples are provided to illustrate the present inventionwithout, however, limiting the same hereto.

EXAMPLE 1 Isolation of the Rhesus MC-5R Gene

A rhesus (M. mulatta) genomic DNA library (Clontech) was screened with arandom primed probe from human MC-5R (Yang et al., 1997, Mol. Endocrinol11: 274-280), identifying one positive lambda phage clone. This clonewas digested with EcoRI and BgIII, analyzed by hybridization, and onepositive fragment (1.6 kb) was subcloned into bluescript SKII (BS SKII).DNA sequencing revealed that EcoRI appears to cleave at a positionimmediately following the start codon, and BgIII cleaves at a positiondown stream of the stop codon. DNA sequencing also revealed a PstI site956 nucleotides down-stream of the EcoRI site. The phage clone was thendigested with PstI which resulted in a 2 kb fragment that hybridized tothe human MC-5R probe. The 2 kb PstI fragment was subcloned intobluescript SKII to determine the 5′ untranslated sequence, confirmingthat the original EcoRI-BgIII fragment encodes the region from the 2ndamino acid to the stop codon. A sense primer,(5′-CCTAGACTCGAGATGAATTCCTCGTTTCAC-3′; SEQ ID NO:3) and an antisenseprimer, (5′-CTCGAGGGATCCTTAATCCCTTCCGGGACAGCT-3′; SEQ ID NO:4) were usedto amplify the open reading frame using the lambda phage clone astemplate. The 1.1 kb fragment encoding the full length rhesus MC-5R wascleaved with XhoI and BamHI and subcloned into pcDNA3.neo for expressionstudies. DNA sequencing confirmed that the sequence in the PCR fragmentwas identical to the previously determined sequence based on the 1.6 kbEcoRI-BgIII fragment and 2 kb PstI fragments.

The deduced nucleotide and amino acid sequence of rhesus MC-5R are shownin FIG. 1 (nucleotide sequence as set forth in SEQ ID NO:1 ) and FIG. 2(amino acid sequence as set forth in SEQ ID NO:2). The first Met residuein the sequence was deduced by determining the upstream nucleotidesequence and locating the presence of a stop codon upstream of the ATGcodon. The rhesus MC-5R receptor protein does not have an unusually longN-terminal sequence as in mouse MC-5R (Fathi et al., 1995, Neurochem.Res. 20: 107-113). As with other melanocortin receptors, severalfeatures in rhMC-5R are shared with other melanocortin receptors and areabsent in most other GPCR's of the rhodopsin sub-family (Fong et al,1996, Cell Signalling 8: 217-224) including the EN motif in TM1, thelack of Cys in the loop between TM2 and TM3 or between TM4 and TM5, theMxxxxxxxY motif in TM5, and the DPxxY motif in TM7. Since allmelanocortin receptors lack Cys residues in the extracellular loops thatare present in other members of the rhodopsin sub-family, interhelicaldisulfide bond (e.g., between the Cys residues near the top of TM3 andTMS) may play the same function as interloop disulfide bond in mostother GPCR's.

EXAMPLE 2 Stable Expression of Rhesus MC-5R

The receptor plasmid was transfected into CHO dhfr- cells usinglipofectamin (GIBCO). Transfected cells were selected by G418 for atleast 2-3 weeks before binding and functional assays were performed. Todetermine the apparent binding affinity of peptides, transfected cellsin 12-well plate were washed once with PBS and binding was done in HBSSwith 25 mM Hepes, 0.5% NaN₃, 0. 1% BSA, and protease inhibitor cocktail(4 ug/ml leupeptin, 40 ug/ml bacitracin, 5 ug/ml aprotinin, 10 uMphospharamidon and 100 uM AEBSF). ¹²⁵I-NDP-α-MSH (Amersham) at aconcentration of 0.1 nM was used and non-specific binding was determinedin the presence of 1 uM unlabeled NDP-α-MSH. After incubation at roomtemperature for 3 hours, wells were washed 3 times by PBS, lysed in 0.5ml of 0.05% SDS and counted by a gamma counter. Alternatively, the sameresults were obtained using cell suspensions followed by GF/C filterfiltration (Huang, et al., 1994, Biochemistry 33: 3007-3013).

To determine the functional activation of receptors, stably transfectedCHO cells were washed once with PBS without Ca⁺⁺/Mg⁺⁺, dissociated bynon-enzymatic dissociation medium (Specialty Media, Inc.) at 37° C. for10 min. Dissociated cells were resuspended in EBSS buffer containing 25mM Hepes, 5 mM MgCl₂, 0.1% BSA and 50 mM IBMX and incubated with agonistat room temperature for 45 min. The agonist activation was terminated byboiling for 4 min. Intracellular cyclic AMP concentration was measuredby the Amersham SPA assay system (Huang, et al., 1997, J. ReceptorSignal Transduc. Res. 17:599-607).

Human MC-5R and MC-5R genomic DNA were obtained from Dr. Ira Gantz(Gantz et al., 1993, J. Biol. Chem. 268: 8246-8250) and subcloned intothe pIRESneo expression vector, and stably transfected into CHO cells.Single clones were used in all pharmacological studies as describedabove.

EXAMPLE 3 Pharmacological Properties of Rhesus MC-5R

A cDNA encoding human MC-5R subcloned into the pIRES1neo expressionvector (Clontech) and stably transfected into CHO cells was used as areference for the pharmacological properties of the rhesus MC-5R. Singleclones were used in all pharmacological studies as described above. Thefunctional properties of rhMC-5R were determined by heterologousexpression in CHO cells. Mock transfected CHO cells did not exhibit anybinding of [¹²⁵I]NDP-α-MSH or any cAMP response to α-MSH.

The rhesus MC-5R was functionally characterized in CHO cells (FIGS. 3Aand 3B). In contrast to the lack of agonist activity of Shu-9119 atMC-5R, Shu-9119 is an agonist for MC-5R. In addition, all testedpeptides are full agonist for the rhesus MC-5R while γ2-MSH and MT-IIare partial agonists for the human MC-5R. As shown in Table 1, manypeptides exhibit a higher binding affinity or activation potency at therhesus MC-5R than at the human MC-5R.

The rhesus MC-5R gene was isolated and purified and the concomitantreceptor was functionally characterized in a heterologous expressionsystem. Shu-9119 is shown to be an agonist for both the rhesus and humanMC-5R. The pharmacological properties of rhesus MC-5R are significantlydifferent from those of the human MC-5R. Especially when the receptoractivation is measured, 5 of the 6 tested peptides are significantlymore potent at the rhesus MC-5R than at the human MC-5R.Species-dependent pharmacological property has been reported (e.g. Fonget al., 1992, J. Biol. Chem. 267: 25668-25671; Oksenberg, et al., 1992,Nature 360: 161-163), apparently due to a small number ofphylogenetically divergent residues in the transmembrane region. Unlikethe NK1 receptor or 5HT1b receptor in which the rank order of potencycan be reversed between two species homologs, the human and rhesus MC-5Rmaintain the same rank order of potency for all tested agonists (FIG.4). The systematic difference in the absolute potency between human andrhesus MC-5R is consistent with the interpretation that the strength ofinteraction (or intermolecular distance) rather than the mode ofinteraction is different between human and rhesus MC-5R. Additionally,the potency of A-MSH and γ2-MSH for the mouse MC-5R (Labbe et al., 1994,Biochemistry 33: 4513-4549) are more similar to those of rhesus MC-5Rand less similar to those of human MC-5R (FIG. 4). Such a pattern wouldpredict that those transmembrane residues that are common in mouse andrhesus MC-5R but different in human MC-5R may determine the observedspecies difference.

TABLE 1 IC50, nM EC50, nM Maximal response (%) Ligands rhMC-5R hMC-5RrhMC-5R hMC-5R rhMC-5R hMC-5R NDP  0.2 ± 0.1 0.5 ± 0.1 0.02 ± 0.01 0.3 ±0.1 100 100 (2) (6) (3) (3) α-MSH 14 ± 1 1800 ± 200  0.2 ± 0.1 12 ± 4 100 100 (2) (4) (3) (3) γ2-MSH 1700 ± 200 9700 ± 2900 17 ± 3  120 ± 25 100 80 (2) (2) (3) (2) MTII   5 ± 0.2 18 ± 3  0.02 ± 0.01 0.8 ± 0.2 10085 (3) (4) (3) (2) Shu9119  0.02 ± 0.001 0.4 ± 0.2 0.007 ± 0.005 0.03 ±0.02 100 100 (3) (4) (2) (3) ACTH1-24 15 ± 1 1050 ± 500  0.2 ± 0.1 5 ± 1100 100 (2) (3) (3) (3)

Table 1 shows that although α-MSH exhibits a very low apparent bindingaffinity for the human MC-5R, its activation potency (EC50=12 nM) ismuch higher. In some brain regions, the α-MSH concentration can reach 4to 54 pmol/g, which is consistent with the notion that α-MSH can be anendogenous agonist for the human MC-5R. The observation that ICS50values are larger than EC50 values actually applies to many MC-5Ragonists (FIG. 4). Based on modeling prediction, activation potency willbe smaller than the intrinsic agonist binding affinity when thereceptor-G protein binding affinity is sufficiently high (Kenakin etal., 1989, Mol. Pharmacol. 35: 214-222), thus implying that MC-5Rcouples to G proteins tightly.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

4 1 1080 DNA rhesus monkey (Macaca mulatta) 1 gctgtctttc tttggtaacctctctggatt gtgaatttaa aatgtgtttt acagtaaatt 60 tgctgccaag acaagaggtgaatttctcca gcaatgaatt cctcgtttca cctgcatttc 120 ttggatctca acctgaatgccacagagggc aacctttcag gacccagtgt cagaaacaag 180 tcttcgccct gtgaaaacatgggcatggct gtggaggtgt ttctcactct gggtgccatc 240 agcctcgtgg agaacatcttggttataggg gccatagtga agaacaaaaa cctgcactgc 300 cccatgtact tcttcgtatgcagcctggcg gtggccgaca tgctggtgag catgtccaac 360 gcctgggaga ccatcaccatctacctgctc aacaacaagc acctagtgat agcagacgcc 420 tttgtgcgcc acattgacaacgtgtttgac tccatgatct gcatttccgt ggtggcgtcc 480 atgtgcagct tgctggccattgcggtggat aggtacgtca ccgtcttcta tgccctgcgc 540 taccaccaca tcatgacggcgaggcgctcg ggggccatca tcgccggcat ctgggctttc 600 tgcaccgggt gcggcatcgtcttcatcctg tactccgagt ccacctacgt catcctgtgc 660 ctcatctcca tgttcttcacgatgctcttc ctcctggtgt gtctgtacat acacatgttc 720 ctcctggcgc ggactcacgccaagcggatg gcggctctgc ccggggccag ctctgcgcgg 780 cagaggacca gcgtgcagggcgcggtcacc ctcaccatgc tgctgggcgt gttcatcgtg 840 tgctgggccc cgttcttcctgcatctcatt ttaatgcttt cttgccctca gaacctctac 900 tgctcttgct tcatgtctcacttcaatatg tacctcatcc tcatcatgtg taactccgtg 960 gtggaccctc tcatatatgccttccgcagc cgggagatgc gcaagacgtt taaggagatt 1020 atttgctgcc gaggcttccgaatcgcctgc agctgtcccg gaagggatta agtacaaagt 1080 2 325 PRT rhesus monkey(Macaca mulatta) 2 Met Asn Ser Ser Phe His Leu His Phe Leu Asp Leu AsnLeu Asn Ala 1 5 10 15 Thr Glu Gly Asn Leu Ser Gly Pro Ser Val Arg AsnLys Ser Ser Pro 20 25 30 Cys Glu Asn Met Gly Met Ala Val Glu Val Phe LeuThr Leu Gly Ala 35 40 45 Ile Ser Leu Val Glu Asn Ile Leu Val Ile Gly AlaIle Val Lys Asn 50 55 60 Lys Asn Leu His Cys Pro Met Tyr Phe Phe Val CysSer Leu Ala Val 65 70 75 80 Ala Asp Met Leu Val Ser Met Ser Asn Ala TrpGlu Thr Ile Thr Ile 85 90 95 Tyr Leu Leu Asn Asn Lys His Leu Val Ile AlaAsp Ala Phe Val Arg 100 105 110 His Ile Asp Asn Val Phe Asp Ser Met IleCys Ile Ser Val Val Ala 115 120 125 Ser Met Cys Ser Leu Leu Ala Ile AlaVal Asp Arg Tyr Val Thr Val 130 135 140 Phe Tyr Ala Leu Arg Tyr His HisIle Met Thr Ala Arg Arg Ser Gly 145 150 155 160 Ala Ile Ile Ala Gly IleTrp Ala Phe Cys Thr Gly Cys Gly Ile Val 165 170 175 Phe Ile Leu Tyr SerGlu Ser Thr Tyr Val Ile Leu Cys Leu Ile Ser 180 185 190 Met Phe Phe ThrMet Leu Phe Leu Leu Val Cys Leu Tyr Ile His Met 195 200 205 Phe Leu LeuAla Arg Thr His Ala Lys Arg Met Ala Ala Leu Pro Gly 210 215 220 Ala SerSer Ala Arg Gln Arg Thr Ser Val Gln Gly Ala Val Thr Leu 225 230 235 240Thr Met Leu Leu Gly Val Phe Ile Val Cys Trp Ala Pro Phe Phe Leu 245 250255 His Leu Ile Leu Met Leu Ser Cys Pro Gln Asn Leu Tyr Cys Ser Cys 260265 270 Phe Met Ser His Phe Asn Met Tyr Leu Ile Leu Ile Met Cys Asn Ser275 280 285 Val Val Asp Pro Leu Ile Tyr Ala Phe Arg Ser Arg Glu Met ArgLys 290 295 300 Thr Phe Lys Glu Ile Ile Cys Cys Arg Gly Phe Arg Ile AlaCys Ser 305 310 315 320 Cys Pro Gly Arg Asp 325 3 30 DNA ArtificialSequence OLIGONUCLEOTIDE 3 cctagactcg agatgaattc ctcgtttcac 30 4 33 DNAArtificial Sequence OLIGONUCLEOTIDE 4 ctcgagggat ccttaatccc ttccgggacagct 33

What is claimed is:
 1. A purified nucleic acid molecule encoding arhesus melanocortin 5 receptor (MC-5R) protein which comprises asequence of nucleotides as set forth in SEQ ID NO:1.
 2. A purified DNAmolecule encoding rhesus melanocortin 5 receptor (MC-5R) protein whereinsaid DNA molecule encodes a protein comprising a sequence of amino acidsas set forth in SEQ ID NO:2.
 3. An expression vector for the expressionof a rhesus MC-5R protein in a recombinant host cell wherein saidexpression vector comprises a DNA molecule which encodes the amino acidsequence of claim
 2. 4. An expression vector of claim 3 which is aeukaryotic expression vector.
 5. An expression vector of claim 3 whichis a prokaryotic expression vector.
 6. An isolated host cell whichexpresses a recombinant rhesus MC-5R protein wherein said isolated hostcell contains the expression vector of claim
 3. 7. An isolated host cellwhich expresses a recombinant rhesus MC-5R protein wherein said isolatedhost cell contains the expression vector of claim
 4. 8. An isolated hostcell which expresses a recombinant rhesus MC-5R protein wherein saidisolated host cell contains the expression vector of claim
 5. 9. Theisolated host cell of claim 6 wherein said rhesus MC-5R protein isoverexpressed from said expression vector.
 10. The isolated host cell ofclaim 7 wherein said rhesus MC-5R protein is overexpressed from saidexpression vector.
 11. The isolated host cell of claim 8 wherein saidrhesus MC-5R protein is overexpressed from said expression vector.
 12. Asubcellular membrane fraction obtained from the isolated host cell ofclaim 9 which contains recombinant rhesus MC-5R protein.
 13. Asubcellular membrane fraction obtained from the isolated host cell ofclaim 10 which contains recombinant rhesus MC-5R protein.
 14. Asubcellular membrane fraction obtained from the isolated host cell ofclaim 11 which contains recombinant rhesus MC-5R protein.
 15. A processfor the expression of a rhesus MC-5R protein in a recombinant host cell,comprising: (a) transfecting the expression vector of claim 3 into asuitable host cell; and, (b) culturing the host cells of step (a) underconditions which allow expression of the rhesus MC-5R protein from theexpression vector.
 16. A purified DNA molecule which consists of asequence of nucleotides as set forth in SEQ ID NO:1.
 17. The purifiedDNA molecule of claim 15 which consists of a nucleotide sequence fromnucleotide 94 to nucleotide 1068 of SEQ ID NO:1.
 18. An expressionvector for the expression of a rhesus MC-5R protein in a recombinanthost cell wherein said expression vector comprises a DNA molecule ofclaim
 17. 19. An expression vector of claim 18 which is a eukaryoticexpression vector.
 20. An expression vector of claim 18 which is aprokaryotic expression vector.
 21. isolated host cell which expresses arecombinant rhesus MC-5R protein wherein said isolated host cellcontains the expression vector of claim
 19. 22. An isolated host cellwhich expresses a recombinant rhesus MC-5R protein wherein said isolatedhost cell contains the expression vector of claim
 20. 23. An isolatedhost cell which expresses a recombinant rhesus MC-5R protein whereinsaid isolated host cell contains the expression vector of claim
 21. 24.A subcellular membrane fraction obtained from the isolated host cell ofclaim 22 which contains recombinant rhesus MC-5R protein.
 25. Asubcellular membrane fraction obtained from the isolated host cell ofclaim 23 which contains recombinant rhesus MC-5R protein.
 26. A purifiednucleic acid molecule encoding a rhesus melanocortin 5 receptor proteinwhich consists of a sequence of amino acids as set forth in SEQ ID NO:2.27. A purified rhesus melanocortin 5 receptor protein which comprises asequence of amino acids as set forth in SEQ ID NO:2.
 28. A purifiedrhesus melanocortin 5 receptor protein which consists of the amino acidsequence as set forth in SEQ ID NO:2.